Proc. R. Soc. B doi:10.1098/rspb.2008.1215 Published online Cheater genotypes in the parthenogenetic ant Pristomyrmex punctatus Shigeto Dobata1,*, Tomonori Sasaki2, Hideaki Mori3, Eisuke Hasegawa4, Masakazu Shimada1 and Kazuki Tsuji2 1Department of General Systems Studies, Graduate School of Arts and Sciences, University of Tokyo, Meguro, Tokyo 153-8902, Japan 2Department of Environmental Sciences and Technology, Faculty of Agriculture, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan 3Department of Ecology and Evolutionary Biology, Graduate School of Life Sciences, University of Tohoku, Aobayama, Sendai 980-8578, Japan 4Laboratory of Animal Ecology, Department of Ecology and Systematics, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan Cooperation is subject to cheating strategies that exploit the benefits of cooperation without paying the fair costs, and it has been a major goal of evolutionary biology to explain the origin and maintenance of cooperation against such cheaters. Here, we report that cheater genotypes indeed coexist in field colonies of a social insect, the parthenogenetic ant Pristomyrmex punctatus. The life history of this species is exceptional, in that there is no reproductive division of labour: all females fulfil both reproduction and cooperative tasks. Previous studies reported sporadic occurrence of larger individuals when compared with their nest-mates. These larger ants lay more eggs and hardly take part in cooperative tasks, resulting in lower fitness of the whole colony. Population genetic analysis showed that at least some of these large- bodied individuals form a genetically distinct lineage, isolated from cooperators by parthenogenesis. A phylogenetic study confirmed that this cheater lineage originated intraspecifically. Coexistence of cheaters and cooperators in this species provides a good model system to investigate the evolution of cooperation in nature. Keywords: evolutionary cheating; Pristomyrmex punctatus; Pristomyrmex pungens; parthenogenesis; social cancer; Emery’s rule 1. INTRODUCTION genotypes coexist with cooperators in natural colonies of Cooperation, as well as competition, is a nearly ubiquitous a social insect, the parthenogenetic ant Pristomyrmex feature of biological systems. Cooperative systems are punctatus (formerly Pristomyrmex pungens; Wang 2003). subject to cheating, which can be defined as an Colonies of social insects provide a typical example of evolutionary strategy that achieves higher individual biological cooperation, which is characterized by a well- fitness than cooperation by exploiting the benefits of developed reproductive division of labour between queens cooperation selfishly without paying the fair costs (on such and workers (Wilson 1971). However, an extraordinary ‘social semantics’, see West et al. 2007a). It has been a life history has evolved in P. punctatus, i.e. the morpho- major goal of evolutionary biologists to explain the logical queen caste is absent and all females are wingless origin and maintenance of cooperation against cheating morphological workers. Furthermore, males are rare (Maynard Smith & Szathma´ry 1995; Keller 1999). and no evidence of sexual reproduction has been found Although numerous theoretical models have been (Itow et al. 1984; Tsuji 1988; T. Sasaki 2001–2005, proposed (for references of ongoing controversies, see personal observation). All monomorphic females are Lehmann & Keller 2006; Taylor & Nowak 2007; West involved in thelytokous reproduction (female-producing et al. 2007b; Wilson 2008) and fascinating experiments parthenogenesis) in their youth and later shift to using micro-organisms have been conducted in the cooperative behaviour, such as colony defence and laboratory (for review, see West et al. 2006), there are foraging, as they age (Tsuji 1990). still a limited number of field studies on cooperation and The characteristically cooperative society summarized cheating. For a thorough understanding of the nature of above seems to be vulnerable to cheating, and this is cooperation, it is important to find tractable systems suggested by previous studies that found unusual individ- uals in some field populations. (figure 1; Itow et al. 1984; containing both cooperators and cheaters under natural Tsuji 1995; Sasaki & Tsuji 2003). Such individuals conditions. Here, we report that distinct cheater (hereafter referred to as the L-type versus normal S-type) are easily distinguished from S-types by their * Author for correspondence ([email protected]). larger body size, ovariole number (four instead of two) and Electronic supplementary material is available at http://dx.doi.org/10. presence of spermathecae (Itow et al. 1984). Behavioural 1098/rspb.2008.1215 or via http://journals.royalsociety.org. analysis revealed that L-types hardly ever take part in Received 29 August 2008 Accepted 23 September 2008 1 This journal is q 2008 The Royal Society 2 S. Dobata et al. Cheater genotypes in an ant species 2001 and 2005, respectively. The mean intracolonial (a) (b) proportion of large workers was 3.5G6.6 and 3.9G6.5%, respectively. Then we counted the number of ocelli for each individual sampled, because males and some L-types are known to have ocelli (whereas the S-type has no ocellus; Itow et al. 1984; Tsuji 1988). Furthermore, we measured the head width (across compound eyes) of the females collected in 2005. (c) Genotypic analysis From the preserved samples, 147 individuals from 17 colonies and 464 individuals from 24 colonies in 2001 and 2005, respectively (570 females and 41 males; 5 S-types and 3–10 L-types per colony in 2001; 10 S-types and 2–10 L-types per colony in the parent generation of 2005; 5 S-types, 1–6 L-types and 1 or 10 males per colony in the offspring generation of 2005), were used for the subsequent Figure 1. (a) S-type and (b) L-type of the parthenogenetic ant genotypic analyses. Total DNA was extracted from the thorax P. punctatus. Scale bar, 1 mm. of each individual using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA) and dissolved in colonial tasks, except reproduction (Sasaki & Tsuji 2003). 100 ml of elution buffer. Therefore, the L-type individuals potentially exploit the cooperative benefit produced by S-types in the production of the next generation. Indeed, Tsuji (1995) investigated (i) Mitochondrial markers field colonies of P. punctatus and showed that an increase As the first screening, a downstream intron region of in the proportion of L-types lowers the reproductive mitochondrial 12S ribosomal RNA gene was amplified for success of nest-mates, which is a symptom of cheating. all samples. A preliminary survey (E. Hasegewa 2005–2006, This trend is confirmed in subsequent field studies unpublished data) had found 4 bp indels in this region among (T. Sasaki, H. Mori, S. Dobata & K. Tsuji 2001–2005, individuals. Polymerase chain reactions (PCRs) were con- unpublished data). ducted using Takara Ex Taq DNA polymerase (Takara Bio, Of particular interest is the genetic background of the Shiga, Japan) and its supplemented buffer system with the primer pair pmf102 (50-CTACATTACTCTATATATAA-30) L-types. To determine whether the L- and S-types share 0 0 genetic interests, we investigated genetic differentiation and pmr101 (5 -AAGATAATAATGAGTTACAGTT-3 ). between these two phenotypes. Our findings indicate that The reaction conditions were 35 cycles of 948C for 30 s, some L-type individuals are genetically distinct from the 458C for 30 s and 608C for 1 min, followed by one cycle of S-type nest-mates, i.e. they represent a lineage that has 728C for 5 min. The amplified fluorescent PCR products specialized in cheating. were analysed using an automated sequencer (CEQ 8000, Beckman & Coulter, Fullerton, CA, USA). 2. MATERIAL AND METHODS (ii) Nuclear microsatellite markers (a) Study species In a preliminary survey, we amplified seven microsatellite Pristomyrmex punctatus is one of the most common ants in markers, Pp1–Pp4 (originally developed for P. punctatus; Japan (Japanese Ant Database Group 2003). Colonies can Hasegawa et al. 2001), L-8, L-15 (Foitzik et al. 1997) and contain up to hundreds of thousands of individuals in their MYRT3 (known to be polymorphic in some ant species; annual life cycles (adults live only 1 year, whereas each colony Bourke et al. 1997), from 25 S-types and 15 L-types collected can last far longer and is potentially immortal; Tsuji 1995). from eight colonies, following the protocols described in each We investigated a population in Kihoku, Mie Prefecture, reference. Polymorphism detection methods were the same as where previous researchers found a colony containing many above. All markers were successfully amplified, but only three L-type individuals (Itow et al. 1984). Twenty-two colonies of them (Pp1, Pp2 and MYRT3) showed polymorphism from one site and 54 colonies from six sites were sampled in among the samples. These three were used for subsequent July 2001 and July 2005, respectively. Each site was amplification and analyses of all sampled individuals. As there approximately 1–5 km apart. From each colony sampled, were a large number of genotypes even within a colony (i.e. some hundreds of adult females were collected and were nest-mates differed in zygosity at the same locus), we carefully placed in pure ethanol. Some colonies contained several considered the results and performed re-genotyping when males in 2005, and these were also collected. necessary. Using these polymorphic microsatellites, the average nest-mate relatedness within each colony was (b) Morphological analysis estimated using the software package RELATEDNESS v. 5.0.8 Based on the number of ovarioles, each of the collected (Goodnight & Queller 2001). females was dissected and classified as the L- or S-type. Some colonies in 2005 contained callow adults, which will over- (iii) Statistics winter and reproduce the next year, and are characterized by First of all, we tested whether the two phenotypes (L- or lipid granules in their abdomens (Tsuji 1995); these callow S-type) differed in the frequency of the multilocus genotypes adults are the offspring of the older adults found in their detected (mitochondrial and nuclear markers combined) colony.